ABSTRACT
To evaluate the inhibitory effects of long antisense RNA on HBV replication in HepG2.2.15 cells. The coding region of HBV S gene was cloned into pTARGET vector in sense and antisense orientations and the recombinant plasmids were transfected into HepG2.2.15 cells which were divided into HBS2 (antisense RNA) group, HBS4 (sense RNA) group and control group. HBsAg and HBeAg in the culture supernate were detected by ELISA. The HBV DNA in the supernate was quantified by real-time PCR. After treatment, the levels of HBsAg in HepG2.2.15 cell supernatants of three groups were 0.621+/-0.027, 3.399+/-0.018 and 2.232+/-0.187 respectively; the levels of HBeAg were 0.749+/-0.019, 1.548+/-0.025 and 1.570+/-0.044 respectively and the levels of HBV DNA were 1.597+/-0.082, 3.381+/-0.297 and 3.610+/-0.063 respectively. The expressions of HBsAg and HBeAg and the HBV DNA level in HBS2 group were remarkably reduced as compared to the control (Z = -2.309, P value is less than 0.05); whereas the sense plasmid transfection (HBS4) did not affect HBeAg (Z = -0.866) and HBV DNA (Z = -1.155) levels in the culture supernate but slightly increased the HBsAg level (Z = -2.309). Antisense RNA might be a useful tool to repress HBV replication.
Subject(s)
Humans , DNA, Viral , Genetics , Gene Expression Regulation, Viral , Genetic Vectors , Hep G2 Cells , Hepatitis B Surface Antigens , Hepatitis B e Antigens , Hepatitis B virus , Genetics , Physiology , Plasmids , RNA Interference , Transfection , Virus Replication , GeneticsABSTRACT
<p><b>OBJECTIVE</b>Major histocompatibility complex class I C-related molecules A and B (MICA and MICB) are innate immune system ligands for the NKG2D receptor expressed by natural killer cells and activated CD8(+)T cells. Our previous study showed that 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, can induce the expression of MICB and sensitized cells to NKL-cell-mediated cytolysis. The aim of this study was to determine the expression level of MICA in HepG2 cells (an HCC cell line) and L02 cells ( a normal liver cell), and to investigate the effect of 5-aza-dC on MICA expression in HepG2 cells.</p><p><b>METHODS</b>Cells were treated with 5-aza-dC, caffeine and ATM-specific siRNA. The cell surface MICA protein on HepG2 cells and L02 cells was determined using flow cytometry. The mRNA level was detected using real time RT-PCR.</p><p><b>RESULTS</b>MICA was undetectable on the surface of L02 cells, but was highly expressed on HepG2 cells. MICA expression was upregulated in response to 5-aza-dC treatment (P less than 0.05), and the upregulation of MICA was partially prevented by pharmacological or genetic inhibition of ataxia telangiectasia mutated (ATM) kinase (P less than 0.05).</p><p><b>CONCLUSION</b>Our data suggest that 5-aza-dC induces the expression of MICA by a DNA damage-dependent mechanism.</p>
Subject(s)
Humans , Ataxia Telangiectasia Mutated Proteins , Azacitidine , Pharmacology , Caffeine , Pharmacology , Carcinoma, Hepatocellular , Metabolism , Cell Cycle Proteins , Metabolism , Cell Line , Cell Membrane , Metabolism , DNA Damage , DNA-Binding Proteins , Metabolism , Flow Cytometry , Hep G2 Cells , Hepatocytes , Metabolism , Histocompatibility Antigens Class I , Genetics , Metabolism , Liver Neoplasms , Metabolism , Protein Serine-Threonine Kinases , Metabolism , RNA, Messenger , Genetics , Metabolism , RNA, Small Interfering , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Tumor Suppressor Proteins , Metabolism , Up-RegulationABSTRACT
<p><b>OBJECTIVE</b>To study the chemical constituents in fresh fleshyscaleaf of Lilium lancifolium.</p><p><b>METHOD</b>The constituents were separated. by various kinds of chromatography and their structures were identified on the basis of spectral analysis.</p><p><b>RESULT</b>Ten compounds were identified regaloside A (1), regaloside C (2), methyl-a-D-mannopyranosid (3), methyl-ca-D-glucopyranoside (4), (25R, 26R) -26-methoxyspirost-5-ene-3p-yl-O-ca-L-rhamnopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->6)]-beta-D-glucopyranoside (5), (25R)-spirost-5-ene-3beta-yl-O-alpha-L-rhamnopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->6)]-beta-D-glucopyranoside (6), (25R, 26R)-17alpha-hydroxy-26-methoxyspirost-5-ene-3beta-yl-O-alpha-L-rhamnopyranosyl-(1-->2)-[beta-D-glucopyra nosyl-(1-->6)]-beta-D-glucopyranoside (7), daucosterol (8), adenoside (9), berberine (10).</p><p><b>CONCLUSION</b>All compounds except 1 and 3 were isolated from this species for the first time, and berberine was first reported in genus Lilium.</p>